• Bulletin of the Korean Chemical Society
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• v.23 no.3
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• pp.481-487
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• 2002

A selective enzyme-linked immunosorbent assay (ELISA) for the insecticide chlorpyrifos was developed. Four haptens for chlorpyrifos were synthesized and two of them were used as immunogens after coupling to keyhole limpet hemocyanin by two differe nt approaches. Rabbits were immunized with either of them and the sera were screened against 4 haptens coupled to ovalbumin (OVA). Using the sera of highest specificity, an antigencoated ELISA was developed, which shows an I50 of 160 ppb with a detection limit of 10 ppb. An antibody-coated ELISA was also developed, which shows an $I_{50}$ of 20 ppb with a detection limit of 0.1 ppb. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except for insecticides chlorpyrifos-methyl and bromophos-ethyl, which makes these assays suitable for the selective detection of chlorpyrifos.

• Bulletin of the Korean Chemical Society
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• v.23 no.4
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• pp.599-603
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• 2002

A selective enzyme-linked immunosorbent assay (ELISA) for the insecticide isofenphos was developed. Three different analogues (haptens) of isofenphos were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group t o use as immunogens or coating antigens. Rabbits were immunized with one of the haptens coupled to BSA for production of polyclonal antibodies and the sera were screened against each of the other two haptens coupled to ovalbumin (OVA). Using the sera of highest specificity, an antigen-coated ELISA was developed, which showed an I50 of 96 ng/mL with the detection limit of 2 ng/mL. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides and the phenol metabolite of isofenphos, which makes the developed assay suitable for the selective detection of isofenphos. An antibody-coated ELISA was also developed, which showed an I50 of 580 ng/mL with a detection limit of 70 ng/mL.

• Parasites, Hosts and Diseases
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• v.33 no.3
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• pp.187-194
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• 1995

The present study aims to observe changing patterns of serum antibody to hleumuvstis calinii in normal rats of different ages and in immunosuppressed rats. The serum IgG antibody was observed by immunoblotting with crude antigen of f carinii which were purified from the lungs of infected rats. The crude antigens separated in SDS-PAGE resolved more than 20 protein bands from 20 to 200 kDa. Of them,40-45, 50-55, 116 and 200 kDa bands were major antigens of R cori.nii. Most of the normal rats of up to 4 weeks had the antibodies reacting the 4 bands, but none of 8-week-old rats revealed the specific antibody. After the rats grew for 40 weeks, all were found to have the antibody in their serum. Same pattern of serum antibody level by age was found in ELISA. When immunosuppressed rats became heavily infected, the antibody in their serum decreased distinctively. The present results suggest that antibodies in normal newborn rats are transferred from their mother and lowered up to 8 weeks. Thereafter, the levels of the antibodies begin to increase by natural exposure to R cnrinii. It was also confirmed that the intensity of P cnrinii infection is inversely related with levels of serum antibodies.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.10-17
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• 2016

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

• Parasites, Hosts and Diseases
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• v.29 no.3
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• pp.293-310
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• 1991

Clonorchis sinensis is a common parasite of man in Korea. Researches on the specific antigens of C. sinensis would be valuable not only because those elucidate the molecular characteristics of this fluke but also because it is applicable to immunodiagnosis. Although many monoclonal antibodies have been used in the field of parasite immunology, few articles on monoclonal antibodies against C. sinensis have been published so far. The aim of this study was to analyse C. sinensis antigens recognized by monoclonal antibodies, and to set up ELISA-inhibition test using C. sinensis specific monoclonal antibodies for improved specificity of immunodiagnostic tests. By fusion between spleen cells of the mice immunized with C. sinensis water-soluble crude adult worm antigens and plasmacytoma cells of mouse origin, 29 hybridoma clones secreting anti-C. sinensis monoclonal antibodies were made, and 8 clones among those were found specific. After cell cloning, isotypes of 6 selected specific monoclonal anti- bodies were determined to be IgGl, IgG2b and IgA. Four exposed antigenic determinants of natural infection were recognized by different specific monoclonal antibodies. By enzyme-immunoelectrotransfer blot, 10 KD, 34 KD antigenic determinants were found to be reacted with CsHyb 0714-20, CsHyb 0605-10 monoclonal antibodies, respectively, The antigenic determinant recognized by CsHyb 0714-20 monoclonal antibody was revealed to be located at the surface and parenchyme of a parasite by indirect immunoauorescent antibody technique, and those reacted with CsHyb 0605-10, CsHyb 0714-25 monoclonal antibodies were found at the parenchyme and intestine. The antigenic determinant reacted with CsHyb 0605-23 monoclonal antibody was found mainly around the uterine eggs. Four antigenic determinants recognized by specific monoclonal antibodies were all found to be present in the early eluted fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration. By conventional ELISA, 75% of clonorchiasis cases were found positive, but 7.1% of normal controls and 37.5% of paragonimiasis cases showed false positives. However, by ELISA-inhibition test using C. sinensis specific monoclonal antibody (CsHyb 0605-23), 77.1% of clonorchiasis cases were found positive, and there were no false positives in normal controls or paragonimiasis cases, indicating 100% specificity. The ELISA- inhibition test using monoclonal antibodies was found to have same sensitivity and definitely high specificity in comparison with conventional ELISA for serodiagnosis of human clonorchiasis.

• Journal of Sensor Science and Technology
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• v.30 no.5
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• pp.337-341
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• 2021

In this paper, we describe the fabrication of a paper-based enzyme-linked immunosorbent assay (ELISA) to detect polygalacturonase (PG), which is used as a biomarker to determine whether a plant is infected with a disease. The proposed paper-based ELISA can analyze the concentration of PG in a short time using a small sample compared to the traditional ELISA, which is generally performed using a well plate. To increase the resolution of the sensor, we optimized the dilution ratio of the HRP-conjugated goat anti-rabbit IgG antibody and the dilution ratio of the anti-PG and HRP-conjugated goat anti-rabbit IgG antibodies. Furthermore, for quantitative analysis of PG concentration, Delta RGB analysis was conducted to detect color changes in the sensing window displayed by the PG samples at various concentrations. Based on the experiment, the fabricated paper-based ELISA could measure at least 0.25 ㎍ of PG and the measurement range was 0.25-2 ㎍. Therefore, the paper-based ELISA for detecting PG is expected to be able to determine the presence or absence of disease in crops at the infection stage in the future.

• Journal of fish pathology
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• v.22 no.2
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• pp.167-172
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• 2009

Antibody-detection enzyme-linked immunosorbent assay (ELISA) was performed to determine whether the absorbance value of ELISA is influenced by the different serum storage conditions of olive flounder Paralichthys olivaceus. Flounder antiserum to bovine serum albumin was stored at -80, -20, 4 and 20${^{\circ}C}$ for 1, 30, 69 and 124 days, respectively. In addition, the flounder antisera were frozen at -80 and -20${^{\circ}C}$, respectively and then repeated 1, 5 and 10 thaw-freeze cycles. No significant difference was shown in ELISA optical density (O.D.) values of sera, which were stored at the above mentioned storage conditions during 124 days. ELISA O.D. values of unfrozen serum samples, which were previously stored at 4${^{\circ}C}$, were almost similar to those of sera undergoing 1, 5, and 10 freeze-thaw cycles after stored at -80 or -20${^{\circ}C}$. In conclusion, the ELISA O.D. values of flounder sera were not affected by various storage conditions: different temperatures (-80, -20, 4 and 20${^{\circ}C}$), durations of storage (1, 30, 69 and 124 days), and repeated thaw-freeze cycles (1, 5, and 10 times).

• Korean Journal of Veterinary Research
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• v.31 no.2
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• pp.223-228
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• 1991

Most progesterone enzyme immunoassays(EIA) are used liquid-phase double-antibody or single-antibody seperation. These methods consume considerable time and reagents because of the requirements for several washing and centrifugation steps involving the reactants. Because of these several problems, we were prompted to develop an effective enzyme-linked immunosorbent assay(ELISA) system that would be equal or superior to RIA for assay of progesterone. The results were obtained as follows. 1. Cross reaction of the progesterone antiserum with other steroids determined was shown with progesterone(100%), $11{\alpha}$-deoxycorti-costerone(2.271%), but the other steroids were shown below 0.9%. 2. Standard curve for progesterone ELISA was shown available difference according to progesterone concentration from 0 to 1,000pg/ml. 3. The lower limit of sensitivity was 0.2pg/well 4. Progesterone concentration was 1.6ng/ml for before parturition, and that was below 0.5ng/ml for after parturition. This development enzyme-linked immunosorbent assay for progesterone can be detected pregnancy diagnosis in cattle, and also applicable 10 research on physiological function including such as reproductive disorders.

• Journal of Applied Biological Chemistry
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• v.48 no.1
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• pp.16-25
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• 2005

To develop an enzyme-linked immunosorbent assay (ELISA) for the detection of the residues of the acaricide fenazaquin, five haptens were synthesized and assessed. A competitive indirect format was used with polyclonal antibodies. Under an optimized condition using the selected rabbit C antiserum, an $IC_{50}$ of $96.97\;ng{\cdot}ml^{-1}$, the detection range of $14.9{\sim}631\;ng{\cdot}ml^{-1}$, and the lowest detection limit of $8\;ng{\cdot}ml^{-1}$ were obtained. Some structurally related compounds of practical use showed low crossreactivities to the antibody. Highest cross-reactivity observed with hapten IV indicates that the antiserum C recognizes very well quinazoline ring, 4-tert-butylphenyl, and an adequate length of spacer arm. The length of spacer arm affected recognition of quinazoline ring and 4-tert-butylphenyl moieties. When applied to apple and pear, recoveries were within acceptable ranges of $93.18{\sim}104.77%$ (n = 4) and $79.40{\sim}111.95%$ (n = 4), respectively.

• Korean Journal of Veterinary Service
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• v.21 no.3
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• pp.255-260
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• 1998

Since bovine leukosis caused considerable economic loss to the dairy industry, seroepidemiologi-cal survey on bovine leukosis was carried out for the dairy herds in Kyunggi province. 1. When compared the results of immunodifussion test with those of enzyme-linked immunosorbent assay(ELISA) for 94 dairy herds sera, the relationship between the immunodifussion test and ELISA were showen high corresponding rate with sensitivity(97.5%) and specificity(92.6%). 2. In immunodiffusion test for bovine leukosis virus (BLV) antibody in 570 dairy cattle from 30 herds, mean positive rate for BLV antibody was 28.2%. The positive rate by districts were 16.5% in central, 35.4% in east, 17.3% in west, 29.1% in south, 31.6% in north, 43.7% in northeast. 3. When the results of serological studies was analyzed by age groups, the number of positive was increased gradually with the advanced in age of herds. The highest positive rate was found in the age over 6 years. 4. Of 30 dairy herds examined, 5 herds(16.7%) have no reactions against BLV antigen while 15 herds (50%) showed the range of 1∼5 positive cattle and 5 herds(16.7%), the rang of over 11 positive cattle.